We propose to continue our studies on the mechanisms of polymerization and its connection to the origin of the pathophysiology of sickle cell disease. We are addressing five issues: 1) Participation of the red cell in nucleation of sickle cell hemoglobin. We have developed a unique capability that has very high precision of determining the effects of membranes on homogeneous nucleation, and will continue our studies on different types of membrane preparation. 2) Mixture Theory for Homogeneous Nucleation: Under-standing the kinetics of mixtures is critical, yet the theory of mixtures has not been expanded to include present models. 3) Identities and strengths of the energetically significant contacts in the polymer. The structure of the polymer is complex. We will measure solubilities for various mutants, as well as solubilities of mixtures of the mutants with HbS. In addition we will measure homogeneous and heterogeneous nucleation rates. From homogeneous nucleation rates and solubilities we can separate the contact energy from the vibrational entropy. Use of mixtures will allow us to ascertain cis-trans issues or inequivalence issues. 4) Tertiary effects on polymerization. This work will study the effect of ligands within the T quaternary structure, with particular emphasis on NOHbS. and 5) Dependence of gel rheology on location within a domain and relation to single fiber behavior. To correlate with single fiber studies, we will make measurements on polymer domains using thermally driven fluctuations. Experiments will use particle tracking methods to compare different regions of polymer domains. Mutants will be used to compare with predicted effects. We will observe the speed with which rigidification propagates through the domain.